Proc. Natl. Acad. Sci. USA Vol. 96, pp. 214–219, January 1999 Medical Sciences NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas YINHUA YU*, FENGJI XU*, HONGQI PENG*, XIANJUN FANG*, SHULEI ZHAO†,YANG LI*, BRUCE CUEVAS*, WEN-LIN KUO‡,JOE W. GRAY‡,MICHAEL SICILIANO§,GORDON B. MILLS*, AND ROBERT C. BAST,JR.*¶ *Division of Medicine and §Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; †Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030; and ‡Department of Laboratory Medicine, University of California, San Francisco, CA 94143 Edited by George F. Vande Woude, National Cancer Institute, Frederick, MD, and approved October 29, 1998 (received for review August 13, 1998) ABSTRACT Using differential display PCR, we have iden- proliferation, depending on cell types. For instance, Rap1 tified a gene [NOEY2, ARHI (designation by the Human Gene positively regulates sustained mitogen-activated protein kinase Nomenclature Committee)] with high homology to ras and rap activation through B-Raf in PC12 cells (12) and induces cell that is expressed consistently in normal ovarian and breast proliferation and transformation in Swiss 3T3 cells (13). Thus, epithelial cells but not in ovarian and breast cancers. Reex- the ras superfamily proteins play an important role in the pression of NOEY2 through transfection suppresses clono- control of cell growth and differentiation. Here, we report a genic growth of breast and ovarian cancer cells. Growth ras-related, maternally imprinted gene mapped to 1p31, which suppression was associated with down-regulation of the cyclin acts as a negative growth regulator in both ovarian and breast y D1 promoter activity and induction of p21WAF1 CIP1.Inan cancers. effort to identify mechanisms leading to NOEY2 silencing in cancer, we found that the gene is expressed monoallelically and is imprinted maternally. Loss of heterozygosity of the MATERIALS AND METHODS gene was detected in 41% of ovarian and breast cancers. In Northern Blot Analysis. NOEY2 cDNA probe was labeled most of cancer samples with loss of heterozygosity, the non- with 32P-dCTP by using a random primer. Total cellular RNA imprinted functional allele was deleted. Thus, NOEY2 appears (15 mg) was separated in 1.2% formaldehyde-agarose gels and to be a putative imprinted tumor suppressor gene whose was immobilized on a Hybond-N1 membrane by standard function is abrogated in ovarian and breast cancers. capillary transfer and UV crosslinking, and then was hybrid- ized to NOEY2 probe by using standard protocol. The mem- Germline abnormalities of several tumor suppressor genes brane was rehybridized to the DNA probe of 18S RNA to have been implicated in hereditary breast and ovarian cancer confirm equal loading among samples. syndromes, including BRCA1, BRCA2 (1, 2), and p53 (3). Transfection and Clonogenic Assays. NOEY2 cDNA [1 However, previously identified genes are silenced or mutated kilobase (kb)] was released from the EcoRI cloning site in the in only a fraction of ovarian and breast cancers; a number of BluescriptyLambda ZapII vector and was inserted into the relevant tumor suppressor genes may not yet have been pcDNA3-neo eukaryotic expression vector (Invitrogen) in identified. Deletions of regions on chromosome 1p have been sense and antisense orientations. The constructs were trans- observed in a variety of human malignancies. 1p31 has been fected into different cell lines by using lipofectamine. After found to have allelic deletion in 28–50% of breast cancers incubation for 48 hours at 37°C, transfected cells were (4–6) and, as described below, in '40% of ovarian cancers. To trypsinized and seeded into 100-mm dishes. Selection medium date, the putative tumor suppressor gene(s) located within this with G418 (400 mgyml to 1000 mgyml) was added. Two weeks region have not been reported. later, colonies were stained by 0.1% Coomassie blue in 30% Gene imprinting, defined as gene expression based on the methanol and 10% acetic acid. gamete of origin, has been implicated in oncogenesis through Luciferase Assay. The vector containing the luciferase re- loss of tumor suppressor gene regulation. The WT1 gene, in porter gene under the control of the human cyclin D1 pro- particular, is part of an imprinted cluster with INS, IGF2, H19, moter (-1745CD1LUC) was provided by R. G. Pestell (Albert and p57KIP2 on chromosome 11p (7). Recent studies have Einstein College of Medicine, New York). NOEY2 sense and suggested that portions of chromosome 1p might also be antisense constructs were cotransfected with the luciferase imprinted. In a subset of neuroblastoma tumors without N-myc reporter, and luciferase activity was measured 48 hr post- amplification, allelic loss of 1p36 is preferentially of maternal transfection as described (14). In control experiments, a vector origin. In contrast, the paternal allele of N-myc was amplified expressing b-galactosidase driven by the CMV promoter was preferentially in those tumors with N-myc amplification (8). cotransfected to ensure comparable transfection efficiency The ras family of protooncogenes is among the most com- between the NOEY2 sense and antisense construct-transfected monly activated in human cancers. Functional activation of the cells (through staining). ras pathway in the absence of genetic mutations has been Western Blot Analysis. The confluent cells in 60-mm culture reported in both breast and ovarian cancer cells (9). Members dishes were lysed by adding boiling SDS sample buffer. The cell of the rap family share high homology with ras proteins and lysates were boiled for an additional 5 min, were passed several have been shown to antagonize ras-mediated activation of times through a 26-gauge needle, and were centrifuged for 5 mitogen-activated protein kinase (10) and cellular transfor- mation in NIH 3T3 fibroblasts (11). Rap proteins also may This paper was submitted directly (Track II) to the Proceedings office. stimulate mitogen-activated protein kinase activation and cell Abbreviations: LOH, loss of heterozygosity; OSE, ovarian surface epithelial; kb, kilobase. The publication costs of this article were defrayed in part by page charge Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. U96750). payment. This article must therefore be hereby marked ‘‘advertisement’’ in ¶To whom reprint requests should be addressed at: Division of accordance with 18 U.S.C. §1734 solely to indicate this fact. Medicine, University of Texas M. D. Anderson Cancer Center, 1515 © 1999 by The National Academy of Sciences 0027-8424y99y96214-6$2.00y0 Holcombe Boulevard, Box 092, Houston, TX 77030. e-mail: rbast@ PNAS is available online at www.pnas.org. notes.mdacc.tmc.edu. 214 Downloaded by guest on September 28, 2021 Medical Sciences: Yu et al. Proc. Natl. Acad. Sci. USA 96 (1999) 215 min. Equal amounts of total cellular protein (15 mg) were NOEY2 cDNA as probe. With this strategy, a full length electrophoresed in 15% SDSyPAGE and were transferred to NOEY2 cDNA sequence and ORF were obtained. The nucle- polyvinylidene fluoride membrane (Millipore). The mem- otide sequence contains a 59 untranslated region of 149 bp, an brane was immunoblotted with anti-NOEY2 mAb (5 mgyml) ORF of 687 bp encoding a 26-kDa protein of 229 amino acids, and was developed by using an ECL system (Amersham). and 660 bp of 39 untranslated sequence with a poly(A) tail. Detection of Loss of Heterozygosity (LOH). One intragenic NOEY2 is a member of the Ras superfamily of small G TA repeat marker was fluorescein isothiocyanate-labeled and proteins. Starting from its N-terminal amino acid 35, NOEY2 was used for PCR-LOH analysis in 3 ovarian cancer cell lines, shares 56% amino acid homology with Rap1A, 56% with 38 primary ovarian cancers, and 8 primary breast cancers Rap1B, 58% with Rap 2A, 62% with Rap2B, 59% with c-K-Ras, and 54% with H-Ras. The NOEY2 gene ORF con- tumor DNA samples paired with normal DNA. DNA fragment y analysis was done by Applied Biosystems 377 Automated tains three motifs typical of Ras Rap family members (21): (i) sequencer. The data were collected automatically and were a highly conserved GTP binding domain, (ii) a putative L NT analyzed by GENESCAN (Applied Biosystems). GENOTYPER II effector domain Y PTIE Y, and (iii) the membrane local- software (Applied Biosystems) was used for allele scoring and izing CAAX motif (where C is cysteine, A is an aliphatic amino assessment of LOH quantitatively according to Canzian’s acid, and X is any amino acid) at the COOH terminus (Fig. 1). NOEY2 formula (15). Within the effector domain, however, differs both PCR–Single-Strand Conformation Polymorphism Sequenc- from Ras and Rap family members where the sequence YDPTIEDSY is found in all Ras and Rap proteins. NOEY2 ing Analysis. The single-strand conformation polymorphism instead has YLPTIENTY. In addition, when compared with analysis was carried out essentially as described (16). After the sequence of p21ras, NOEY2 has substitutions of alanine for single-strand conformation polymorphism analysis of the cod- glycine at amino acid 12 and glycine for glutamine at amino ing region, PCR fragments with abnormal mobility were 1 acid 61, consistent with constitutive activation of a small G cloned into the PCR-Script Amp SK( ) cloning vector (Strat- protein. agene). More than 10 clones of each sample were sequenced Loss of NOEY2 Expression in Ovarian and Breast Carci- by using an Applied Biosystems PRISM 377 DNA automatic nomas. An NOEY2 message of 1.9 kb was detected by North- sequencer and a Big Dye terminator cycle sequencing kit. Upstream promoter region sequences (1.9 kb) were amplified by four pairs of primers with a 221M13 tail. Purified PCR products were directly sequenced by using a 21M13 Dye primer cycle sequencing kit or a Big Dye terminator cycle sequencing kit (Applied Biosystems).